Project Summary Relatively little is known about the functional distinctions and coordination of roles of the cell types comprising the resident dendritic cell (DC) network in the human lung. The CD1c+ DC subtype has been implicated in cellular immunity to influenza A virus (IAV), which is important for clinical outcome, heterosubtypic broad IAV immunity and immunity in the elderly. We propose to elucidate the mechanisms underlying the immunological responses to IAV of the CD1c+ primary DC subtype. In concert with mathematical modeling in Project III, we will test the hypothesis that the programmatic responses and maturation state of the CD1c+ DC during IAV infection emerges from the interplay of factors including differences among single cells, differences among individual infectious particles as well as among IAV strains, and the effects of CD1c+ crosstalk and that originating from other lung cells during infection mediated by the secreted factor microenvironment. In Aim 1, we will study the mechanisms of single cell response variation to IAV infection in primary circulating CD1c+ DC using single cell assays including mass cytometry, FISH during flow cytometry, and single cell RNA sequencing. In Aim 2, we will investigate mechanisms of primary circulating CD1c+ DC responses to infection by seasonal and pandemic IAV using a panel of wild-type and recombinant H1N1 IAV. In Aim 3, we will investigate how the microenvironment modulates the early responses to IAV infection in experiments with fresh human lung tissue and circulating CD1c+ DC. We expect this work to provide insight into the complex integrated immunity to IAV initiated in the lung that will further the rational development process for treatment of IAV infections and improved immunity to mucosal vaccines.